Microfluidic devices for culturing primary mammalian neurons at low densities
Microfluidic devices for culturing primary mammalian neurons at low densities
复制标题
DOI：
10.1039/b705266a
复制
发表时间：
2007-01-01
期刊：
影响因子：
6.1
通讯作者：
Gillette, Martha U.
中科院分区：
文献类型：
--
作者：
Millet, Larry J.;Stewart, Matthew E.;Gillette, Martha U.
微流体装置已用于研究多种细胞类型的高密度培养物。然而，由于细胞间信号传导是局部的，因此需要开发能够维持少量神经元并能够分析微环境的培养系统。这种培养物很难维持稳定的形式，并且在使用原代哺乳动物神经元时很难防止细胞死亡。我们证明，来自大鼠的出生后原代海马神经元可以在使用聚二甲基硅氧烷（PDMS）制造的纳升体积微型装置中以低密度培养。这样做需要额外的制造步骤，即用多种溶剂对 PDMS 进行连续萃取/洗涤，从而去除未交联的低聚物、溶剂和用于固化聚合物的铂催化剂残留物。我们发现此步骤显着提高了 PDMS 设备的生物相容性。虽然神经元在开放通道微装置中存活 >= 7 天，但在由未经处理的天然 PDMS 制成的闭路装置中培养神经元的能力仅限于 = 7 天。与高压灭菌的 PDMS 和天然的、未经处理的 PDMS 进行比较表明，溶剂处理的聚合物在维持培养物中原代神经元的低密度方面具有优越性。当直接观察神经元对局部基质的亲和力时，我们发现轴突定位于通道角，并且在混合设备中更喜欢 PDMS 表面而不是玻璃。当通过重力流向通道灌注介质时，培养的海马神经元存活 >= 11 天。提取 PDMS 可以提高微流体装置的生物相容性，从而能够研究可识别神经元的分化和局部细胞外信号的表征。
Microfluidic devices have been used to study high-density cultures of many cell types. Because cell-to-cell signaling is local, however, there exists a need to develop culture systems that sustain small numbers of neurons and enable analyses of the microenvironments. Such cultures are hard to maintain in stable form, and it is difficult to prevent cell death when using primary mammalian neurons. We demonstrate that postnatal primary hippocampal neurons from rat can be cultured at low densities within nanoliter-volume microdevices fabricated using polydimethylsiloxane (PDMS). Doing so requires an additional fabrication step, serial extractions/washes of PDMS with several solvents, which removes uncrosslinked oligomers, solvent and residues of the platinum catalyst used to cure the polymer. We found this step improves the biocompatibility of the PDMS devices significantly. Whereas neurons survive for >= 7 days in open channel microdevices, the ability to culture neurons in closed-channel devices made of untreated, native PDMS is limited to = 7 days. Comparisons made to autoclaved PDMS and native, untreated PDMS reveal that the solvent-treated polymer is superior in sustaining low densities of primary neurons in culture. When neuronal affinity for local substrates is observed directly, we find that axons localize to channel corners and prefer PDMS surfaces to glass in hybrid devices. When perfusing the channels with media by gravity flow, cultured hippocampal neurons survive for >= 11 days. Extracting PDMS improves biocompatibility of microfluidic devices and thus enables the study of differentiation of identifiable neurons and the characterization of local extracellular signals.